Saturation mutagenesis reveals the importance of residues alpha R145 and alpha F146 of penicillin acylase in the synthesis of beta-lactam antibiotics Jager, Simon A. W.; Shapovalova, Irina V.; Jekel, Peter A.; Alkema, Wynand B. L.; Svedas, Vytas K.; Janssen, Dick B.; Švedas, Vytas K. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. AbstractPenicillin acylase (PA) from Escherichia coli can catalyze the coupling of an acyl group to penicillin-and cephalosporin-derived ␤-lactam nuclei, a conversion that can be used for the industrial synthesis of ␤-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues ␣R145 and ␣F146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5-15-fold. Moreover, due to increased tendency of the acyl-enzyme intermediate to react with ␤-lactam nucleophile instead of water, mutants ␣R145G, ␣R145S and ␣R145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic ␤-lactam antibiotics.

Penicillin acylase (PA) from Escherichia coli can catalyze the acylation of 6-aminopenicillanic acid (6-APA), a conversion that is applicable in the biocatalytic preparation of semi-synthetic ␤-lactam antibiotics such as ampicillin. The efficiency of this kinetically controlled conversion, in which an amide or ester acts as the acyl donor, is dependent on the kinetic properties of the enzyme. To further improve the synthetic properties of PAs, family gene shuffling was performed with the PA-encoding genes of the PAs from E. coli, Kluyvera cryocrescens and Providencia rettgeri. Of these three PAs, the E. coli enzyme possessed the best properties for the synthesis of ampicillin. Shuffled recombinant libraries were pre-screened for activity by growth selection, followed by testing the catalytic performance in ampicillin synthesis using HPLC. Three clones with improved synthetic properties were selected and sequence analysis showed that the shuffled genes were hybrids of the PA-encoding genes from E. coli and K. cryocrescens, with additional point mutations. The hybrid enzymes displayed a 40-90% increase in the relative rate of acyl transfer to the ␤-lactam nucleus during ampicillin synthesis. This increase was not accompanied by a reduction of synthetic activity that has previously been reported for mutants of E. coli PA constructed by site-directed mutagenesis. Similar improvements in acyl transfer were obtained for the synthesis of amoxicillin, cephalexin and cefadroxil, making the new hybrid enzymes interesting candidates for the biocatalytic synthesis of several ␤-lactam antibiotics.

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